PROJECT ABSTRACT Despite evidence that chemokines are increased in OA joints, few studies have examined their role in OA and the mechanism by which they promote joint destruction. The goal of this proposal is to determine if the CC- chemokine receptor 2 (CCR2) is a critical mediator of joint damage and pain in OA. Our preliminary studies and recent work by others suggest CCR2 inhibition may reduce both structural damage and pain in OA. If this is true, it would be a major breakthrough in the management of OA. However, more work is needed to confirm these findings and determine the optimal time to inhibit CCR2 during the OA process. Our data from a community- based cohort study of human OA shows that higher baseline serum levels of human CCL2 (which binds CCR2 receptor) correlates with progression of radiographic OA at 5-years follow up. Using the destabilized medial meniscus (DMM) murine model of injury-induced OA, we found that expression of CCL12 (the functional homologue of hCCL2), a CCR2 ligand we have previously found to be critical in joint development, increases in articular cartilage and bone during early OA. OA changes in articular cartilage (cartilage loss, increased MMP13 levels) and bone (bone sclerosis, osteophytes) and pain behavior were ameliorated by systemic blockade of the CCR2, if given for 4wks total within the first 8wks after DMM. However, if treatment was maintained for 8wks or more it was less effective on subsequent structural progression while still inhibiting pain, suggesting a disconnect between structure and pain that has been noted in human OA. These data suggest that CCR2 signaling has cellular and stage specific roles in OA, with beneficial effects when targeted at early stages. We hypothesize that injury-induced alterations in joint mechanics upregulate the CCR2 pathway in cartilage and bone in a time- dependent manner altering the structural organization of both tissues, ultimately leading to OA and pain. We propose to determine the role of dysregulated CCR2 signaling in cartilage at sequential times during injury- and age-induced OA, in order to establish the contribution of this tissue to the whole joint degeneration and OA pain (Aim 1). We will also evaluate whether osteoblast expression of CCR2 contributes to joint damage and pain during injury-induced OA (Aim 2). To accomplish this, we will obtain an inducible tissue-specific CCR2 deletion in chondrocytes (Aim 1) and osteoblasts (Aim 2) by combining chondrocyte- or osteoblast-specific expression of CreER with Tam injections in CCR2flox/flox mice crossed with Aggrecan-CreERT2 or Col1?1CreER, respectively. Because the myeloid infiltration consisting of macrophages and osteoclasts may cause accelerated OA bone remodeling that may reflect on bone damage and, indirectly, on cartilage homeostasis, we will also conditionally inactivate CCR2 in macropahges/osteoclasts (Aim 3) by crossing CCR2flox/flox mice with LysMERCre. We will also evaluate potential pathways altered by each tissue-selective CCR2 deletions. In addition, by using in-vitro human primary chondrocyte cultures, we will study the signaling pathways by which activation of CCR2 with its multiple ligands stimulates MMP production.